Production of a Vitamin C precursor using genetically modified organisms

ABSTRACT

An enzyme for conversion of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonic acid (2-KLG) and a genetically modified organism that expresses all the fermentation enzymes needed to convert glucose to 2-KLG (a precursor to ascorbic acid) using the new enzyme are described. Preferably, the organism is Erwinia citreus, or a mutated strain of Erwinia citreus, unable to use 2,5-DKG or 2-KLG as a sole carbon source, into which the gene for a 2,5-DKG reductase, produced by Corynebacterium sp., SHS 752001, has been inserted. The preferred transformed organism expresses the fermentation enzymes Erwinia citreus normally expresses for fermentation of glucose to 2,5-DKG and, in addition, an enzyme Corynebacterium sp. SHS 752001 expresses for fermentation of 2,5-DKG to 2-KLG.

TECHNICAL FIELD OF THE INVENTION

This invention relates to DNA sequences, recombinant DNA molecules,organisms containing such sequences and molecules, the expression ofcertain enzymes by such organisms, and the production, by fermentation,of a Vitamin C precursor using such organisms and enzymes. Morespecifically, this invention relates to an expression vehicle andgenetically modified organisms, transformed by that vehicle, thatexpress enzymes used to convert glucose, or another carbon source, byfermentation to 2-keto-L-gluconic acid (2-KLG), a chemical precursor toVitamin C (ascorbic acid).

BACKGROUND ART

There are several processes for producing Vitamin C. One processinvolves a number of chemical synthesis steps and one fermentation step.Briefly, the steps are hydrogenation of glucose to sorbitol,fermentation of sorbitol to sorbose using Acetobacter suboxydans,sorbose acetonization, diacetone sorbose oxidation to 2-KLG,esterification of 2-KLG, and conversion of the ester to ascorbic acid.This process is complex and requires a relatively high capitalinvestment for an operating plant.

Another process involves two fermentation steps. The process starts withfermentation of glucose to 2,5-diketo-D-gluconate (2,5-DKG) by Erwiniasp.; fermentation of 2,5-DKG to 2-KLG by Corynebacterium sp.;esterification of 2-KLG; and conversion of the ester to ascorbic acid.One study has shown that D-gluconate and 2-keto-D-gluconate (2-KDG) areproduced sequentially from glucose by Erwinia sp. before 2,5-DKG isproduced in the first fermentation step. See T. Sonoyama et al.,"Production of 2-keto-L-gulonic acid from D-glucose by Two-StageFermentation," App. and Envir. Microbiol., 43, 1064-69 (1982). Thistwo-step fermentation process, although having a somewhat lower capitalcost than the Acetobacter process, is still complex and expensive tooperate.

Still another process for converting glucose to 2-KLG is referred to inEuropean patent application No. 132,308. That application refers to theconversion of glucose to 2-KLG in a single step fermentation process. Itfirst refers to Corynebacterium sp. ATCC 31090 as a source of a DNAsequence coding for a particular 2,5-DKG reductase (an enzyme that issaid to catalyze the fermentation of 2,5-DKG to 2-KLG). This DNAsequence, with its own or a synthetic ribosome binding site, is thensaid to be inserted "downstream" of an E. coli trp or tac promoter orthe pACYC184 CAT promoter in an expression vector. The vector is alsosaid to contain a gene coding for tetracycline resistance or otherselectable marker, and an origin of replication derived from plasmidsColE1, 115A, or RSF 1010. A host cell, Erwinia herbicola (ATCC 21998),is then said to be transformed with the vector. On fermentation thistransformed cell is said to produce 2-KLG from glucose in one step. Theconversion of glucose to 2-KLG in that process, however, is not fullysatisfactory because the yield of 2-KLG is very low and the time offermentation to obtain even that low yield is too long.

Accordingly, a single organism capable of converting a carbon source,such as glucose, to 2-KLG at acceptable rates and in a singlefermentation step is still a goal that has not been attained.

SUMMARY OF THE INVENTION

The present invention solves the problem of finding a single organismcapable of converting glucose, or other carbon source, into 2-KLGquickly and in high yield. In one embodiment, this invention provides anexpression vehicle capable of transforming a host so that it performsall of the fermentation steps required for converting glucose, or othercarbon source, to 2-KLG in a single fermentation at acceptableconversion rates, without intermediate product recovery or intermediatepurification steps. The 2-KLG resulting from practice of the presentinvention may then be esterified and converted to ascorbic acid (VitaminC), as in the conventional processes described above.

In contrast to the process of the present invention, the knowncommercial fermentation processes for converting glucose to 2-KLGrequire two separate organisms, for example strains of Erwinia andCorynebacterium, to transform glucose to 2-KLG.

One advantage of the present invention is that a single strain of agenetically modified organism achieves significant yields of 2-KLGdirectly from glucose in a single fermentation. Thus, the ability of theprocess of this invention to use a single fermentation step results in arelatively simpler process than the known commercial process so thatless process equipment and less energy is required to produce Vitamin Cfrom glucose.

Another object of the present invention is to provide a novel 2,5 DKGreductase and a novel transformed organism superior to those referred toin European patent application No. 132,308, and the processes andproducts of the present invention are accordingly unexpectedly improvedand patentable over the processes and products of European patentapplication No. 132,308.

Still other objects and aspects of the invention will be apparent fromthe specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a partial amino acid sequence from the N-terminus of the2,5-DKG reductase of this invention;

FIG. 2 depicts part of the amino acid sequence of a 14,000d molecularweight cyanogen bromide fragment of the 2,5-DKG reductase of thisinvention;

FIGS. 3a-e depict the sequences of several nucleotide probes used tolocate by hybridization portions of the Corynebacterium genomecontaining the 2,5-DKG reductase gene of this invention; and

FIG. 4 depicts the DNA sequence of the 2,5-DKG reductase gene of thisinvention and the corresponding amino acid sequence of the 2,5-DKGreductase of this invention.

FIG. 5 depicts the construction of plasmid pPLred332 from plasmids 210*and pcBR13. In FIG. 5, the symbols have the following meanings: PL,P_(L) promoter; S-D, Shine-Dalgarno sequence; ori, origin ofreplication; Lac, lac promoter; amp-r, ampicillin resistance gene.

BEST MODE OF CARRYING OUT THE INVENTION

In order that the present invention may be more fully understood, thefollowing detailed description is provided. In this specification someof the following terms are employed:

Nucleotide

A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), aphosphate, and a nitrogenous heterocyclic base. The base is linked tothe sugar moiety via the glycosidic carbon (1' carbon of the pentose)and that combination of base and sugar is called a nucleoside. The basecharacterizes the nucleotide. The four DNA bases are adenine ("A"),guanine ("G"), cytosine ("C"), and thymine ("T"). The four RNA bases areA, G, C, and uracil ("U"). For DNA, "P" indicates either of the purines(A or G), "Q" indicates either of the pyrimidines (C or T), and "N"indicates any of the four bases (A, G, C, or T). For RNA, "P," "Q," and"N" have the same meanings except that "U" is substituted for "T."

DNA Sequence

A linear array of deoxy nucleotides connected one to the other byphosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.

Codon

A DNA sequence of three nucleotides (a triplet) that encodes, throughits mRNA, an amino acid, a translation start signal, or a translationtermination signal. For example, the nucleotide triplets TTA, TTG, CTT,CTC, CTA, and CTG encode for the amino acid leucine ("Leu"); TAG, TAA,and TGA are translation stop signals; and ATG is a translation startsignal that also codes for methionine. .

Reading Frame

The grouping of codons during the translation of mRNA into amino acidsequences. During translation the proper reading frame must bemaintained. For example, the DNA sequence GCT GGT TGT AAG may beexpressed in three reading frames or phases, each of which produces adifferent amino acid sequence:

GCT GGT TGT AAG--Ala-Gly-Cys-Lys

G CTG GTT GTA AG--Leu-Val-Val

GC TGG TTG TAA G--Trp-Leu-(STOP)

Polypeptide

A linear array of amino acids connected one to another by peptide bondsbetween the a-amino and carboxy groups of adjacent amino acids. When"polypeptide" is used in this specification, it will be understood bythose skilled in the art to include the term "protein."

Genome

The entire DNA of a cell or a virus. It includes, inter alia, the DNAcoding for the polypeptides of the cell and operator, promoter, andribosome binding and interaction sequences, including sequences such asthe Shine-Dalgarno sequences for each of those coding sequences.

Gene

A DNA sequence that encodes through its template or messenger RNA("mRNA") a sequence of amino acids characteristic of a specificpolypeptide.

Expression

The process undergone by a gene to produce a polypeptide. It includestranscription of the DNA sequence to a mRNA sequence and translation ofthe mRNA sequence into a polypeptide.

Plasmid

A non-chromosomal double-stranded DNA sequence comprising an intact"replicon" such that the plasmid is replicated in a host cell. When theplasmid is placed within a unicellular organism, the characteristics ofthat organism may be changed or transformed as a result of the DNA ofthe plasmid. For example, a plasmid carrying a gene for tetracyclineresistance (Tet^(R)) transforms a cell previously sensitive totetracycline into one which is resistant to it. A cell transformed by aplasmid is called a "transformant."

Phage or Bacteriophage

A bacterial virus. Many phages consist of DNA sequences encapsulated inprotein envelopes or coats ("capsids").

Cloning Vehicle

A plasmid, phage DNA, or other DNA sequence that is able to replicate ina host cell. A cloning vehicle is characterized by one or a small numberof endonuclease recognition sites at which such DNA sequences may be cutin a determinable fashion without attendant loss of an essentialbiological function of the DNA, e.g., replication, production of coatproteins, or loss of promoter or binding sites. A cloning vehicleusually contains a marker suitable for use in the identification oftransformed cells, e.g., tetracycline resistance or ampicillinresistance. A cloning vehicle is often called a vector.

Cloning

The process of obtaining a population of organisms or DNA sequencesderived from one such organism or sequence by asexual reproduction.

Recombinant DNA Molecule or Hybrid DNA

A molecule, comprising segments of DNA from different genomes joinedend-to-end outside of living cells, that may be maintained in livingcells.

Expression Control Sequence

A sequence of nucleotides that controls and regulates expression ofgenes when operatively linked to those genes. They include the lacsystem, the β-lactamase system, the trp system, the tac system, the trcsystems, the major operator and promoter regions of phage λ, the controlregion of fd coat protein, the early and late promoters of SV40,promoters derived from polyoma virus and adenovirus, metallothioninepromoters, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells andtheir viruses or combinations thereof. For mammalian cells the gene canbe linked to an eukaryotic promoter such as that for the SV40 earlyregion coupled to the gene encoding dihydrofolate reductase andselectively amplified in Chinese hamster ovary cells to produce a cellline containing many copies of actively transcribed eukaryotic genes.

In one embodiment this invention is directed to recombinant DNAmolecules, taken from Corynebac-terium sp. SHS 752001, characterized bya DNA sequence that codes for the 2,5-DKG reductase of this invention.In another embodiment, this invention is directed to a host, preferablyErwinia citreus, transformed by such a recombinant DNA molecule.

The recombinant DNA molecules of this invention are characterized by aDNA seguence coding for the 2,5-DKG reductase of this invention and anexpression control sequence that is operatively linked to that DNAsequence in the recombinant DNA molecule. A wide variety of expressioncontrol sequences may be used in the recombinant DNA molecules of thisinvention. These include the lac system, the β-lactamase system, the trpsystem, the tac system, the trc systems, the major operator and promoterregions of phage λ, the control region of fd coat protein, the early andlate promoters of SV40, promoters derived from polyoma virus andadenovirus, metallothionine promoters, the promoter for3-phosphoglycerate kinase and other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, neomycin phosphotransferase promoter, and other sequences knownto control the expression of genes of prokaryotic or eukaryotic cellsand their viruses or combinations thereof. For mammalian cells, the genecan be linked to an eukaryotic promoter such as that for the SV40 earlyregion coupled to the gene PG,10 encoding dihydrofolate reductase andselectively amplified in Chinese hamster ovary cells to produce a cellline containing many copies of it. The preferred expression controlsequence of this invention is derived from the lac sequence of pUC8.

In addition, the recombinant DNA molecules of this invention maycomprise DNA sequences from a variety of plasmids and phages that allowthem to replicate in the chosen host. Preferably, they also include aselection marker, e.g., a DNA sequence coding for a drug resistance.Such plasmid and phage sequences may be derived from, for example,segments of chromosomal, non-chromosomal, and synthetic DNA sequences,such as various known derivatives of SV40 and known bacterial plasmids,e.g., plasmids from E. coli including col E1, pCR1, pBR322, pMB9 andtheir derivatives, wider host range plasmids, e.g., RP4, phage DNAs,e.g., the numerous derivative of phage λ, e.g., NM989, and other DNAphages, e.g., M13 and Filamenteous single-stranded DNA phages andvectors derived from combinations of plasmids and phage DNAs, such asplasmids which have been modified to employ phage DNA or otherexpression control sequences or yeast plasmids such as the 2 μ plasmidor derivatives thereof.

It should of course be understood that not all vectors and expressioncontrol sequences will function in the same way to express the modifiedDNA sequences of this invention and to produce the new 2,5-DKG reductaseof the present invention. Neither will all hosts function equally wellwith the same expression system. One skilled in the art, however, maymake a selection among these vectors, expression control sequences, andhosts without undue experimentation and without departing from the scopeof this invention. For example, in selecting a vector, the host must beconsidered because the vector must replicate in it. The vector's copynumber, the ability to control that copy number, and the expression ofany other proteins encoded by the vector, such as antibiotic markers,should also be considered.

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe system, its controllability, and its compatibility with the DNAsequence encoding the 2,5-DKG reductase of the present invention,particularly as regards potential secondary structures.

The DNA sequence for the 2,5-DKG reductase of this invention may be usedto produce that reductase in a wide variety of hosts, e.g., bacteriasuch as strains of E. coli, such as E. coli C600, E. coli ED8767, E.coli DH1, E. coli LE392, E. coli HB101 , E. coli X1776, E. coli X2282,E. coli MRCI, and strains of Pseudomonas, Bacillus and Streptomyces,yeasts and other fungi, animal hosts, such as Chinese hamster ovarycells or mouse cells, other animal (including human) hosts, plant cellsin culture or other hosts. After expression, the enzyme is then usefulfor transforming 2,5-DKG to 2-KLG.

Hosts for the DNA sequence of the 2,5-DKG reductase of this inventiongenerally should be selected by consideration of their compatibilitywith the chosen vector, the toxicity of the 2,5-DKG reductase of thepresent invention to the host, susceptibility of the desired protein toproteolytic degradation by host cell enzymes, contamination or bindingof the 2,5-DKG reductase by host cell proteins difficult to removeduring purification, expression characteristics, the ability of the hostto produce and secrete the 2,5-DKG reductase, the ability of the host tofold the reductase correctly, the fermentation requirements of the host,the ease of purification of the 2,5-DKG reductase from the host, safety,and cost.

In one embodiment, Erwinia citreus SHS 2003 is selected as a hostbecause it produces 2,5-DKG from glucose, and 2-KLG may be produceddirectly from glucose by a transformed Erwinia citreus SHS 2003.

Most preferably hosts of the present invention are Erwinia citreus,SHS2003 (Ferm-P No. 5449; ATCC No. 31623) and a strain mutated fromErwinia citreus SHS2003, Erwinia citreus ER1026, that is unable to useeither 2,5-DKG or 2-KLG as a sole carbon source.

The following example shows some embodiments of the invention but is notintended to limit the scope of the invention.

EXAMPLE

Identification Of A Polypeptide Sequence of the 2,5-DKG Reductase OfThis Invention, and Preparation of Cloning Vectors

Corynebacterium sp. SHS752001 was described in T. Sonoyama et al.,"Production of 2-keto-L-gulonic acid from D-glucose by two-stagefermentation,"App. and Envir. Microbiol., 43, 1064-69 (1982). Thus, thedisclosed strain of Corynebacterium, selected as the donor of a DNAsequence coding for a 2,5-DKG reductase. To aid in identifying the2,5-DKG reductase gene from that strain of Corynebacterium, a sample ofan enzyme was isolated and purified to 95% purity using the followingprocedure:

A. Cultivation of Corynebacterium SHS 752001

1. A freeze-dried culture of Corynebacterium sp. SHS 752001 wasrehydrated immediately after opening by adding 0.4 ml of 0.9% NaCl(sterilized at 120° C. for 20 minutes) to the contents of a vialcontaining the freeze-dried culture;

2. The culture was transferred to a test tube containing 8 mls of asolution containing 0.5% glucose, 0.5% yeast extract (Difco), 0.5%peptone (Difco), 0.1% KH₂ Po₄, 0.02% MgSO₄.7H₂ O and 2.0% agar. Thesolution pH was 7.0, and the solution had been sterilized at 120° C. for15 min. Forty hours after the addition of the culture at 28° C., theculture was suspended with 0.4 ml of 0.9% NaCl that had been sterilizedat 120° C. for 20 min.;

3. Five lots of 0.1 ml of the suspension from step 2 were added to fiveagar slants containing the same ingredients as the solution of step 2.The cultures were fermented for the same amount of time and under thesame conditions as step 2. The cultures were suspended with 5 ml of 0.9%NaCl (sterilized at 120° C. for 20 min.), and 2.5 ml of the suspensionwas transferred to a each of ten seed flasks containing 500 ml ofmedium.

4. A solution containing: 1% glucose, 0.5% yeast extract (Difco), 0.5%peptone (Difco), 0.1% NaNO₃, 0.1% KH₂ PO₄, and 0.02% MgSO₄.7H₂ O, andhaving a pH of 7.0 was sterilized at 120° C. for 15 minutes. Sixty ml ofthe solution were added to a 500 ml flask containing the culture. After16-18 hours of cultivation at 28° C., the culture content of 10 such 500ml flasks were transferred to a 30 liter (L) jar fermentor; and

5. Twenty liters of a solution at pH 7.2, containing 1.8% glucose. 2.7%corn steep liquor 0.31% NaNO₃, 0.06% KH₂ PO₄, 4.4 ppm ZnSO₄ 7H₂ O, 0.72ppm MnCl₂ -4H₂ O, 0.2 ppm Vitamin B₁ -HCl, 0.15 ppm Calciumpanthothenate, and 0.005% antiform (Adekanol), was added to the 30 literjar fermentor containing the culture. The fermentor was incubated at 28°C. with agitation at 400 rpm and an air flow rate of 0.5 v.v.m.. Thefermentation was stopped when glucose disappeared from the culture (22h). The final pH was 7.5 and the final OD was 19.2.

B. Preparation of cell extract

About 750 gr of cells were harvested from the 30 liter jar fermentor bycentrifugation using a Sharples centrifuge (10,000 G, 10 minutes) Thecells were suspended in 0.1 M tris-HCl buffer (pH 7, 2.5 L), washedthree times with the same buffer (2.5 L in each case) using acentrifuge, and finally resuspended in 1.6 L of the buffer (OD 150). Thecells (as 80 ml of cell suspension) were disrupted by sonication (160watts for 7 min.). Unbroken cells and debris were removed bycentrifugation (15,000 G for 30 min.), and the supernatant (1 L) waspooled.

C. Fractionation by AmSO₄

The protein material that precipitated between 40% and 70% saturationwas collected by centrifugation, and redissolved in 80 mls of 0.1Mtris-HCl buffer at pH 7. The solution was dialyzed against 0.02 Mtris-HCl buffer at pH 7 overnight.

D. Ion-Exchange Chromatography

The dialyzed solution (99 ml) was placed on a DEAE-Sepharose CL-6Bcolumn (1.6×30 cm) previously equilibrated with 0.02 M tris-HCl buffer(pH7). The column was washed stepwise with 0.02 M tris-HCl buffercontaining zero and 0.2 M NaCl (pH 7). The enzyme was eluted with thesame buffer containing 0.3 M NaCl (pH 7). The active fractions werepooled and the protein was concentrated by adding AmSO₄ up to 70%saturation. The precipitate was collected and dialyzed as in step C.

E. First Affinity Chromatography

The dialyzed solution (37 ml) obtained from step D was placed on anAmicon Matrix Red A column (1.6×19 cm) previously equilibrated with 0.02M tris-HCl buffer (pH 7). The column was washed stepwise with 0.02Mtris-HCl buffer (pH 7) containing 0.3-0.5M NaCl. The enzyme was elutedwith the same buffer containing 0.7-1.0M NaCl. The active fractions (90ml) were pooled.

F. Second Affinity Chromatography

0.02M tris-HCl buffer (pH 7, 225 ml) was added to the pooled fractionfrom step E, and the resulting solution was placed on an Amicon MatrixRed A column (1.9×12.3 cm) previously equilibrated with 0.02M tris-HClbuffer (pH 7). The column was washed with 0.02M tris-HCl buffer (pH 7)containing 0.2M NaCl. The enzyme was eluted with the same buffercontaining 0.5 mM NADPH. The active fractions (35 ml) were pooled andconcentrated by ultra-filtration (cut-off below M.W. of 10,000) toremove NADPH.

To demonstrate that the 2,5- DKG reductase of this invention was notdenatured during the sonication process and to confirm that the 2,5-DKGreductase of this invention converts 2,5-DKG to 2-KLG, a mixture of 0.1Mtris-HCl (pH7) was prepared containing 7 mg NADPH, 2 mg 2,5-DKG and 50μl cell sonicate at a total protein concentration of 2.5 mg/μl. Thetotal reaction volume was 200 μl. The products of the reaction wereanalyzed by HPLC, and the presence of 2-KLG was confirmed.

Antibodies to the 2,5-DKG reductase of this invention were prepared. Theantibodies were developed by injecting a rabbit intradermally atmultiple sites with 100 μg of the 2,5-DKG reductase of this invention inFreunds adjuvant and boosting with 50 μg of the enzyme in Freundsincomplete solution twenty-one days later. Serum was taken ten daysafter the boost injection and was shown to be positive for anti-(2,5-DKGreductase of this invention) activity in an enzyme-linkedimmunoabsorbant ("ELISA") assay.

The purified enzyme was sequenced from the N-terminus using a highsensitivity gas phase sequenator manufactured by Applied Biosystems. Thepartial sequence obtained by this method is shown in FIG. 1. A questionmark in the Figure indicates that a particular amino acid could not bedetermined with absolute certainty.

The purified enzyme was also cleaved using a standard cyanogen bromideclevage procedure. A portion of the amino acid sequence of one 14,000dfragment produced by that method is shown in FIG. 2.

In order to select from a library of clones a DNA sequence coding forthat 2,5-DKG reductase of this invention, a series of oligonucleotideprobes (shown in FIGS. 3a-e) was prepared, using the phosphotriestermethod. These probes were derived from the amino acid sequences of FIGS.1 and 2.

Because of the degeneracy of the genetic code, each probe was actually afamily of structurally related molecules. For example, the 14-mer DNAprobe of FIG. 3a (Probe I of FIG. 1) had a redundancy of 96 with one C-Tmis-match over the predicted sequence, the 14-mer DNA probe of FIG. 3b(Probe II of FIG. 1) had a redundancy of 32 with one G-T mis-match, the17-mer DNA probes of FIGS. 3c and 3d (Probe III of FIG. 1) each had aredundancy of 32 and differed from one another only in the first twopositions, and the 14-mer DNA probe of FIG. 3e (Probe IV of FIG. 2) hada redundancy of 32.

To construct libraries of Corynebacterium DNA to permit screening by theprobes of FIGS. 3(a-e) to select the 2,5-DKG reductase gene of thisinvention, Corynebacterium sp SHS 752001 was lysed by treating a cellsuspension of Corynebacterium sp. SHS 752001 with lysozyme (1 mg/l in 10mM Tris-HCl (pH 8); 1 mM EDTA and 20% (w/v) sucrose followed by sodiumdodecylsulfate (SDs) (5 mg/ml). DNA from the lysed Corynebacterium sp.SHS 752001 cells was then fragmented using the restriction endonucleaseSau3a in a buffer comprising 150 mM NaCl, 6 mM Tris-HCl (pH 7.9), 6 mMMgCl₂, and 100 μg/ml bovine serum albumin. The DNA fragments were theninserted into pUC8 vectors at a BamHI site using the method of J. Vieraand J. R. Messing, Gene, 19, 259 (1982), and the recombinant plasmidswere transformed into Escherichia coli JM83, W3110i^(q), and W3110i^(q)recA. The method for transformation of the recombinant plasmids into E.coli JM83 is set forth in J. R. Messing, R. Corea, P. H. Seeburg,Nucleic Acids Res., 9, 309 (1981). Similar methods were used for otherhost strains.

Colonies of the resulting library were screened with the probes of FIGS.3(a-e) using the procedure set forth in Wallace, R. B., Johnson, M. J.,Hirose, T., Miyake, T., Kawashima, E. H. and Itakura, K., Nucleic AcidsRes., 9, 879-94 (1981). The probes were hybridized at 37° C. and werealso washed at 37° C. in a standard wash comprising 6×SSC, 5×Denhardt'sbuffer, 0.1% SDS, and 0 μg/ml t-RNA. Of seventeen colonies that werepositive (hybridized) with the probe of FIG. 3e, two were also positivewith the probes of FIGS. 3c or 3d or both. The two recombinant plasmidsof those clones were designated pCBR10 and pCBR13. The foreign DNA ofeach of the two recombinant plasmids was 3.0 kb long.

Properties of Expression Vectors and Transformants According To theInvention

In order to determine the properties of the recombinant plasmids,including production rates of the 2,5-DKG reductase of this invention bythe transformed hosts, the two recombinant plasmids and pUC8, a vectornot containing the DNA coding for the 2,5-DKG reductase of thisinvention, were transformed into E. coli W3110i^(q) recA and Erwiniapunctata by the procedure set forth in Cohen, S. N., Chang, A. C. Y.,and Hsu, L., Proc. Natl. Acad. Sci. U.S.A., 69, 2110 (1972)). VectorpUC8, which is related to pBR322, has a strong lac promoter locatedslightly upstream from a BamH1 site.

Several procedures were examined for releasing the 2,5-DKG reductase ofthis invention from the transformed cells that produced it so thatproduction rates could be measured. Sonication was found to be best forE. coli and Erwinia.

Extracts of E. punctata and of E. coli carrying pCBR10 and pCBR13 wereanalyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) (Laemmli, U. K. Nature, 227, 680-85 (1970)) and by Westernblotting (Thomas, P. S., Proc. Natl. Acad. Sci. U.S.A., 77, 5201-05(1980)) using anti-(2,5-DKG reductase of this invention) antibody,produced above, to determine the amount of 2,5-DKG reductase of thisinvention produced by the transformants. Extracts of E. coli W3110i^(q)recA (pCBR13) treated with the lac inducer isopropylβ-D-thiogalactopyranoside (IPTG) contained about five times more enzymethan extracts from uninduced cells with the plasmid carrying the gene.

Confirmation that the 2,5-DKG Reductase of this Invention was notAltered During Recombination

The 2,5-DKG reductase produced by E. punctata and E. coli aftertransformation with pCBR10 and pCBR13 was examined to see if it was thesame enzyme isolated from Corynebacterium sp. SHS 752001.

The molecular weight of the protein, labelled by western blotting ofextracts of Erwinia (pCBR13), of E. coli W3110i^(q) recA (pCBR13), andof Corynebacterium, was the same as the molecular weight of the purified2,5-DKG reductase of this invention (i.e., about 29,000d). Based oncomparison of the blots for the different cells, it was estimated that1-2% of the total cell protein of Erwinia (pCBR13) was the 2,5-DKGreductase of this invention.

We have made further constructions using the λP_(L) promoter, a strongpromoter which can be controlled by the λcI repressor protein.Expression of the λP_(L) promoter can be controlled by changing thetemperature from 30° C. to 42° C. in a strain which also carries theλcI₈₅₇ temperature-sensitive repressor. Increased yields of 2-KLG fromrecombinant strains were obtained using this promoter system.

FIG. 5 shows the physical map of the vector used for making the plasmidsexpressing 2,5-DKG reductase under the control of the P_(L) promoter.Plasmid pPLred332 was made by inserting the fragment coding for 2,5-DKGreductase from plasmid pCBR13 into plasmid p210*. The fragment insertedwas generated by digestion of pCBR13 with restriction endonucleasesEcoRI and HindIII; the two fragments were separated by electrophoresisthrough low melting agarose. The vector was also digested with EcoRI andHindIII (see FIG. 5) so that the appropriate fragments could be ligated.The resulting plasmid, pPLred332, was transformed into strain W3110cI_(ts) to form strain EC1083. This strain carries a chromosomalinsertion of the lambda temperature-sensitive repressor gene cI857Plasmid pPLred332, which comprises the vector element derived from 210*and the insert from pCBR13 as shown in FIG. 5, specifies 2,5-DKGreductase which has the same molecular weight, in both E. coli and inErwinia citreus, as that specified by pCBR13.

Erwinia citreus ER1026 was transformed with pPLred332 to form strainER1116.

In order to confirm the amino acid and DNA sequence of the 2,5-DKGreductase of this invention, the 3 kb insert of pCBR13 was sequenced bythe Maxam and Gilbert technique (Proc. Natl. Acad. Sci. U.S.A., 74, 560(1977) and Maxam, A. M. and Gilbert, W., Meth. Enzym., 65, 499-560(1980)) and by Sanger sequencing using plasmid M13 (Sanger, F.,Nickelen, S. and Coulsen, A. R., Proc. Nat'l Acad. Sci. U.S.A., 74,5463-67 (1977)). Examination of the sequence of the 3.0 kb insertrevealed a sequence that coded almost exactly for the first sixty aminoacids determined for the enzyme itself (as shown in FIG. 1). The 3.0 kbinsert also contained a sequence that coded almost exactly for the aminoacid sequence of FIG. 2. The end of the reductase gene in the 3.0 kbinsert was indicated by a STOP codon. In this way the reductase gene wasdetermined to be 831 nucleotides long (omitting the STOP codon andincluding ATG, the START codon), and its sequence is shown in FIG. 4.

The DNA sequence coding for the amino acid sequence of the 2,5-DKGreductase of this invention may be compared to the DNA sequence codingfor, and the amino acid sequence of, the polypeptide of European PatentApplication No. 132,308. FIG. 4 shows both the DNA sequence and aminoacid sequence for the 2,5-DKG reductase of this invention. FIG. 4 ofEuropean Patent Application No. 132,308 shows the purported DNA sequenceand amino acid sequence of that polypeptide. A comparison of the twofigures clearly shows that the 2,5-DKG reductase of this invention andits DNA sequence are markedly different from the amino acid sequence andDNA sequence of European Patent Application No. 132,308.

The Michaelis constant (Km) of an enzyme measures the kinetics of thatenzyme. The lower the value of the constant, the higher the activity, orvelocity, of the enzyme. The Michaelis constant for the 2,5 DKG reportedin European Application No. 132,308 is 15.5 mM, and the Michaelisconstant for the 2,5-DKG reductase of this invention (using 100 μM NADPHas a cofactor) in 0.1 M Tris-HCl, pH 7.0 at 30° C. is 2.0 mM.

The following procedures illustrate fermentation of glucose to 2-KLG, aVitamin C precursor, using a genetically modified organism of thisinvention.

Fermentation of Glucose to 2-KLG Using Transformed Erwinia

Erwinia citreus SHS 2003, on deposit with the American Type CultureCollection, Maryland, U.S.A., ATCC No. 31623 and on deposit with theFermentation Research Institute, Yatabe, Japan, FERM-P No. 5449,normally expresses enzymes for converting glucose to 2,5-DKG. Thisstrain was transformed with pCBR13, which, as described above, containsa gene that codes for the 2,5-DKG reductase of this invention, using theprocedure set forth in Cohen, S. N., Chang, A. C. Y., and Hsu, L., Proc.Nat'l. Acad. Sci. U.S.A., 69, 2110 (1972). The resulting strain,designated ER817, thus should contain the genes for all the enzymesrequired for converting glucose to 2-KLG in a single fermentation.

Strain ER817 was inoculated onto a plate of L-broth agar that containedampicillin (40 μg/ml). The strain had been taken from a stock culturekept in L-broth plus 15% glycerol at -70° C. After the plate hadundergone 24 hours incubation at 18° C., 10 ml of a seed medium(glycerol, 5 g/l; corn steep liquor, 27.5 g/l; KH₂ PO₄, 1 g/l; pH 6.8)in a 250 ml conical flask was inoculated to an absorbance at 650 mm of0.05 (A₆₅₀ =0.05) with strain ER817 taken from the plate.

The resulting seed culture was incubated for 24 hours at 18° C. withrotary shaking. Ten ml of a production medium (corn steep liquor, 30g/l; KH₂ PO₄, 1 g/l; NaCl, 1 g/l; CaCO₃, 29 g/l; glucose, 10 g/l; pH6.8) in a 250 ml conical flask was inoculated to A₆₅₀ =0.2 with the seedculture and was incubated with rotary shaking at 28° C. for 65 hours.

The culture was then centrifuged and the supernatant was analyzed forthe presence of 2-KLG by high performance liquid chromatography (HPLC).Fifty μl samples of the supernatant were analyzed using a Biorad HPX-87Hcolumn at 65° C. (0.6 ml/min) in 0.18N H₂ SO₄. A peak having a retentiontime of 8.86 min indicates the presence of 2-KLG. The retention times ofthe other compounds of interest are as follows: 2,5-DKG, 8.46 min;2-keto-D-gluconic acid (2-KDG), 9.20 min; gluconic acid, 10.0 min; andfucose, 12.1 min.

The various samples of supernatant showed a peak at 8.86 min, indicatingthe presence of 2-KLG at a concentration of 1 g/l. To confirm that thecompound producing a peak at 8.86 min was 2-KLG, a quantity of known2-KLG (as the sodium salt) was added to a sample of supernatant. Thatincreased only the peak at 8.86 min, thereby providing the confirmationsought. In contrast, when a sample of culture was taken only 18 hoursafter inoculation (rather than after 65 hours), HPLC analysis of thesupernatant contained 2,5-DKG in a concentration of 7 g/l but nodetectable 2-KLG.

To confirm further that 2-KLG (and not 2-KDG) was actually beingproduced in the 65-hour culture, a quantity of known 2-KDG (as thecalcium salt) was added to a sample of the supernatant to aconcentration of 1 g/l. HPLC analysis of that spiked culture then showedthe presence of a new peak at 9.16 min, but essentially no change in the2-KLG peak at 8.86 min.

Another method of demonstrating the production of 2-KLG by the culturewas also used. By converting any 2-KLG produced by the fermentation toascorbic acid, a reducing agent, the reducing capacity of a fermentationmedium containing 2-KLG may be measured, and the amount of 2-KLG presentin the solution before its conversion to ascorbic acid may becalculated. To compensate for the presence of reducing agents other thanascorbic acid in a fermentation mixture, a first sample of afermentation mixture is treated to convert 2-KLG to ascorbic acid andthen the total reducing capacity of the sample is determined. A secondsample is prepared that, after conversion of the 2-KLG, has had allascorbic acid eliminated from the medium. The reducing activities of thetwo samples are then compared, and the difference in activity shouldindicate the amount of ascorbic acid present in the samples, and hencethe 2-KLG present in the fermentation medium.

A sample of supernatant of a fermentation medium was treated to convertany 2-KLG present into Vitamin C. 75 pl of 8N HCl were added to 50 μl ofthe supernatant and the mixture was incubated for 30 min at 95° C. 120μl of 5N NaOH were added and the pH was adjusted to 3.75 using 1N sodiumacetate.

Ascorbic acid and any other reducing agents in the mixture would reducethe tetrazolium salt MTT[3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] in thepresence of the electron carrier PMS [5-methylphenazium methylsulphate]to a formazan. Such treatment was carried out and it indicated the totalof all reducing agents (including Vitamin C) in the mixture.

To facilitate specific determination of Vitamin C, a sample of thefermentation mixture before treatment with MTT and PMS was treated withascorbic acid oxidase and oxygen to destroy any Vitamin C present.Subsequent treatment with MTT and PMS then indicated the amount ofreducing agents other than Vitamin C present, and by difference theamount of Vitamin C (and thus of 2-KLG) was determined. In this way thepresence of Vitamin C in the mixture and, therefore, the presence of2-KLG in the supernatant were proven.

Using the procedure for converting 2-KLG to Vitamin C with samplescontaining known concentrations of 2-KLG allowed preparation of astandard curve. The curve showed that the supernatant obtained from the65-hour culture of strain ER817 contained 1 g/l of 2-KLG, agreeing withresults of the HPLC analyses. 2-KLG was not detected in the supernatantof a similarly produced and treated culture of another strain of Erwiniacitreus that lacks pCBR13 (strain SHS2003).

Conversion of glucose to 2-KLG was carried out with the followingfermentation procedure using transformed strain ER817. Inoculation wascarried out as in Example 1 but incubation in the production medium wasfor 30 hours instead of for 65 hours and 100 ml of culture were used.The culture supernatant contained 1 g/l of 2-KLG but less than 0.1 g/lof glucose, 2-KDG, 2,5-DKG, or gluconic acid.

One way of obtaining better yields of 2-KLG is to ensure that none ofthe intermediates produced in the fermentation of glucose to 2-KLG arebeing consumed in biological reactions that are unrelated to 2-KLGproduction. Accordingly, a mutant of Erwinia citreus (SHS2003) that wasunable to use 2,5-DKG or 2-KLG as sole carbon source was isolatedfollowing nitrosoguanidine mutagenesis in the procedure described byMiller. Experiments in Molecular Genetics (mold Spring Harbor, 1974),except that the cells were incubated in the presence of nitrosoguanidineat 30° C. After mutagenesis, the cells were incubated for 18 h at 30° C.in M9 medium, that contained glucose (0.2% w/v) or glycerol (0.4% w/v)as described in Miller, Experiments in Molecular Genetics (Cold SpringHarbor, 1974). Samples of the culture were spread onto plates of M9glucose or M9 glycerol medium and were then replicated onto plates of M9medium containing 2,5-DKG (0.5% w/v) or 2-KLG (0.2% w/v). Mutants unableto grow on either of these media were purified by restreaking and weretested for their ability to use a variety of substrates as sole carbonsources. Several mutants were obtained that could not use any of2,5-DKG, 2-KDG or 2-KLG as sole carbon sources. One such mutant wastransformed with plasmid pCBR13 to form strain ER1037, which was thentested for its ability to convert glucose to 2-KLG.

A culture of Erwinia citreus ER1037 was deposited in the DeutschSammlung von Mikroorganismen culture collection. The culture wasdeposited on July 22, 1985 and is identified as follows: DSM No. 3404.

A culture of strain ER 1037 was grown for 18 h in L-broth and 90 ml ofthis culture was inoculated into 500 ml of a medium comprising (perliter): K₂ HPO₄, 4 g; KH₂ PO₄, 1 g; NH₄ Cl, 1 g; CaCl₂, 0.01 g; K₂ SO₄,2.6 g; casamino acids, 10 g; yeast extract, 1.5 g; corn steep liquor, 10g; D-mannitol, 20 g; and glucose, 10 g. After 20 h growth at pH 6.0 in a1 liter fermenter (aeration at 0.7 vessel volumes min⁻¹ (v.v.m.);agitation at 800 r.p.m.), the concentration of 2-KLG in the growthmedium was 6.25 g/liter.

Using Erwinia citreus ER1026 transformed with the plasmid pPLred332, afurther improvement in yield of 2-KLG can be achieved. The resultingstrain, ER1116, is grown as described above. However, instead ofterminating the fermentation described in the example after 20 h, thefermentation was extended by the addition of a further 10 g/L of glucose12 h after inoculation.

Two further additions of 10 g/L of glucose can also be made 36 h and 60h after the start of the fermentation. A final level of 2-KLG in thefermentation broth was 19.83 g/L representing a conversion from glucoseof 49.4%.

It will be apparent to those skilled in the art that variousmodifications may be made in the invention without departing from itsscope or spirit, and our basic construction can be altered to provideother embodiments which utilize the processes and compositions of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the claims appended hereto rather than thespecific embodiments which have been presented as examples.

We claim:
 1. A DNA sequence coding for a 2,5-DKG reductase, said DNAsequence selected from the group consisting of:(a) a synthetic orisolated DNA sequence of the formula: ##STR1## (b) synthetic or isolatedDNA sequences which, as a result of the degeneracy of the genetic code,code for a polypeptide encoded by the foregoing DNA sequence.
 2. Aprocess for producing 2-KLG from glucose comprising the steps of:(a)isolating a mutant of Erwinia unable to use 2,5-DKG as the sole carbonsource; (b) transforming said mutant with a recombinant DNA moleculeComprising a DNA sequence according to claim 1 operatively linked to alambda P_(L) promoter; and (c) fermenting said mutant in a mediumcomprising glucose.
 3. The process of claim 2 wherein the pH of themedium is maintained at about pH 6.0.
 4. A recombinant DNA moleculecomprising a DNA sequence coding for a 2,5-DKG reductase, said DNAsequence selected from the group consisting of:(a) a DNA sequence of theformula: ##STR2## (b) DNA sequences which, as a result of the degeneracyof the genetic code, code for a polypeptide encoded by the foregoing DNAsequence.
 5. The recombinant DNA molecule of claim 4, wherein said DNAsequence is operatively linked to an expression control sequence in themolecule.
 6. The recombinant DNA molecule of claim 5, wherein saidexpression control sequence is selected from the group consisting of thelac system; the β-lactamase system; the trp system; the tac system; thetrc system, the major operator and promoter motor regions of phage λ;and the control region of fd coat protein, the early and late promotersof SV40, promoters derived from polyoma virus and adenovirus,metallothiomine promoters, the promoter for 3 -phosphoglycerate kinaseor other glycolytic enzymes, the promoters of acid phosphatase, e.g.,Pho5, the promoters of the yeast α-mating factors; promoters formammalian cells such as the SV40 early and late promoters, adenoviruslate promoter, and other sequences that control the expression of genesof prokaryotic or eukaryotic cells and their viruses and combinationsthereof.
 7. A transformant comprising a host transformed with at leastone recombinant DNA molecule according to claim 5 or
 6. 8. Thetransformant of claim 7, wherein said host makes 2,5-diketo-D-gluconicacid.
 9. The transformant of claim 8, wherein said host belongs to thegenus Erwinia.
 10. The transformant of claim 9, wherein the host isErwinia citreus.
 11. The transformant of claim 6, wherein said host isselected from the group consisting of Erwinia citreus SHS2003 andErwinia citreus ER1026.
 12. A method for producing a 2,5-DKG reductasecomprising culturing a host transformed by a recombinant DNA moleculeaccording to claim 4 or
 2. 13. The method of claim 12, furthercomprising the step of isolating said 2,5-DKG reductase.
 14. A processfor producing 2-KLG from glucose comprising the step of fermenting thetransformant of claim 10 in a medium comprising glucose.
 15. A processfor producing vitamin C, comprising the steps of:(a) fermenting thetransformant of claim 10 in a medium comprising glucose to produce2-KLG; and (b) converting said 2-KLG to vitamin C.
 16. The recombinantDNA molecule of claim 15 wherein said molecule is pCBR13.